12  Gene Enrichment Analysis III

Removed Ribosomal &Mitochondria-Related Genes from DESeq2 Results

To identify biological pathways and processes affected by Cre-mediated ApoE4→ApoE2 conversion in liver tissue, we performed complementary enrichment analyses using the high-confidence differentially expressed genes from Method 1 (combined batches, no batch adjustment).

Removed 16 mitochondrial genes and 146 ribosomal genes from the dataset.

12.0.1 Gene Set Enrichment Analysis (GSEA)

pathway	NES	padj	size
GOBP_MITOCHONDRIAL_RESPIRATORY_CHAIN_COMPLEX_ASSEMBLY	2.056	0.018	96

Gene Set Enrichment Analysis (GSEA) of the complete ranked gene list, excluding mitochondrial and ribosomal genes, identified enrichment of the mitochondrial respiratory chain complex assembly in the liver upon ApoE2 switch (NES = 2.056, padj = 0.018).

There are 50 leading-edge genes driving the enrichment of the GOBP_MITOCHONDRIAL_RESPIRATORY_CHAIN_COMPLEX_ASSEMBLY, including Ndufb4b, Pet117, Ndufb4c, Ndufb1, Ndufb3, Ndufb8, Ndufaf2, Lyrm2, Ndufs4, Pet100, among others._All these genes are nuclear-encoded mitochondrial proteins involved in electron transport and ATP synthesis_ and are upregulated in liver following ApoE4→ApoE2 conversion.

12.1 Brain

12.1.1 Gene Set Enrichment Analysis (GSEA)

pathway	NES	padj	size
GOBP_EPOXYGENASE_P450_PATHWAY	2.236	0.030	26
GOBP_REGULATION_OF_CHROMATIN_ORGANIZATION	2.218	0.013	35
GOBP_ARACHIDONIC_ACID_METABOLIC_PROCESS	2.210	0.011	59
GOBP_XENOBIOTIC_METABOLIC_PROCESS	2.139	0.006	98
GOBP_SPLICEOSOMAL_COMPLEX_ASSEMBLY	2.121	0.029	49
GOBP_POSITIVE_REGULATION_OF_MRNA_CATABOLIC_PROCESS	1.889	0.038	91
GOBP_CELLULAR_RESPONSE_TO_XENOBIOTIC_STIMULUS	1.860	0.029	153
GOBP_POSITIVE_REGULATION_OF_MRNA_METABOLIC_PROCESS	1.855	0.045	125
GOBP_DENDRITE_MORPHOGENESIS	1.854	0.011	188
GOBP_ACTION_POTENTIAL	1.815	0.013	160
GOBP_REGULATION_OF_MRNA_METABOLIC_PROCESS	1.813	0.006	274
GOBP_RESPONSE_TO_ALCOHOL	1.778	0.045	164
GOBP_DENDRITE_DEVELOPMENT	1.711	0.010	315
GOBP_SYNAPSE_ASSEMBLY	1.609	0.045	267
GOBP_CHROMATIN_REMODELING	1.519	0.010	472
GOBP_MRNA_PROCESSING	1.460	0.044	453
GOBP_OXIDATIVE_PHOSPHORYLATION	-2.060	0.045	124

GSEA of brain tissue revealed 17 significantly enriched pathways.

12.1.1.1 Reducing Redundant Pathways with collapsePathways

Because many pathways shared the same leading-edge genes, we used the collapsePathways function from the fgsea package to remove redundancy and isolate distinct biological themes. This method groups pathways with overlapping gene sets and keeps only the most significantly enriched representative from each cluster, ensuring that the analysis reflects independent biological processes rather than repetitive signals.

pathway	NES	padj	size
GOBP_REGULATION_OF_CHROMATIN_ORGANIZATION	2.218	0.013	35
GOBP_ARACHIDONIC_ACID_METABOLIC_PROCESS	2.210	0.011	59
GOBP_SPLICEOSOMAL_COMPLEX_ASSEMBLY	2.121	0.029	49
GOBP_CELLULAR_RESPONSE_TO_XENOBIOTIC_STIMULUS	1.860	0.029	153
GOBP_ACTION_POTENTIAL	1.815	0.013	160
GOBP_REGULATION_OF_MRNA_METABOLIC_PROCESS	1.813	0.006	274
GOBP_RESPONSE_TO_ALCOHOL	1.778	0.045	164
GOBP_DENDRITE_DEVELOPMENT	1.711	0.010	315
GOBP_SYNAPSE_ASSEMBLY	1.609	0.045	267
GOBP_CHROMATIN_REMODELING	1.519	0.010	472
GOBP_OXIDATIVE_PHOSPHORYLATION	-2.060	0.045	124

This analysis identified 11 representative pathways that summarize the main biological processes affected by hepatic ApoE2 expression. The refined pathway set highlighted significant changes in chromatin organization, lipid metabolism, synaptic remodeling, and mitochondrial function in brain tissue.

Notably, genes related to oxidative phosphorylation showed reduced expression in the brain, in contrast to their increase in the liver, indicating a distinct metabolic adjustment driven by altered ApoE signaling.

There are 74 leading-edge genes driving the enrichment of the GOBP_MITOCHONDRIAL_RESPIRATORY_CHAIN_COMPLEX_ASSEMBLY, including Ndufs7, Cox7a1, Atp5f1e, Cox6a1, Cox4i2, Cox8a, Ndufa3, Ndufa7, Ndufa11, Cox7a2, among others._All these genes encode mitochondrial proteins involved in electron transport and ATP synthesis_ and are downregulated in the brain following ApoE4→ApoE2 conversion.

12.2 Common Leading-Edge Genes in Liver and Brain

Leading-edge analysis revealed several mitochondrial genes as main contributors to pathway enrichment in both liver and brain. To pinpoint shared molecular mechanisms underlying the effects of ApoE isoform conversion, we compared the overlapping leading-edge genes between the two tissues.

This analysis identified 12 mitochondrial genes commonly altered in both liver and brain following ApoE4→ApoE2 conversion: Uqcc2, Ndufb4, Ndufb9, Ndufb2, Ndufs5, Ndufs8, Ndufb6, Ndufs6, Ndufb8, Ndufb7, Sdhaf2, Ndufb5

tissue	gene	log2FoldChange	padj
brain	Ndufb2	-0.433	0.000
liver	Ndufb2	0.294	0.055
brain	Ndufb4	-0.503	0.000
liver	Ndufb4	0.561	0.001
brain	Ndufb5	-0.211	0.000
liver	Ndufb5	0.259	0.002
brain	Ndufb6	-0.344	0.000
liver	Ndufb6	0.384	0.000
brain	Ndufb7	-0.256	0.000
liver	Ndufb7	0.341	0.004
brain	Ndufb8	-0.270	0.000
liver	Ndufb8	0.765	0.000
brain	Ndufb9	-0.465	0.000
liver	Ndufb9	0.379	0.016
brain	Ndufs5	-0.417	0.000
liver	Ndufs5	0.267	0.055
brain	Ndufs6	-0.341	0.000
liver	Ndufs6	0.346	0.005
brain	Ndufs8	-0.384	0.000
liver	Ndufs8	0.280	0.004
brain	Sdhaf2	-0.212	0.000
liver	Sdhaf2	0.254	0.015
brain	Uqcc2	-0.545	0.000
liver	Uqcc2	0.386	0.030

These genes showed opposite patterns of regulation, increasing in the liver while decreasing in the brain. This contrast indicates that hepatic ApoE2 expression influences mitochondrial function in a tissue-specific manner, likely shaping the broader metabolic adjustments observed in this model.

12.3 Analysis of Brain metabolic Genes

To understand how hepatic ApoE2 expression influences brain energy metabolism, we analyzed genes involved in glucose transport, glycolysis, lactate shuttling, glycogen turnover, and mitochondrial function. Most genes in these pathways showed modest but consistent shifts, revealing a coordinated metabolic adjustment rather than isolated changes.

log2FoldChange indicates differential expression in brain following hepatic ApoE4→ApoE2 conversion (i.e., ApoE2 effect).

12.3.1 Glycolysis Core Genes

Several glycolytic enzymes, including Aldoa, Pgk1, Eno1, Ldha, and Aldoc, showed reduced expression, suggesting a slower glycolytic flux. In contrast, Hk1 increased, indicating stronger glucose trapping within cells. Together, these changes suggest a transition from rapid anaerobic glycolysis toward more regulated and efficient glucose utilization.

gene	log2FoldChange	padj
Ldhb	-0.272	0.000
Aldoa	-0.354	0.000
Pgk1	-0.209	0.000
Eno1	-0.261	0.000
Aldoc	-0.237	0.002
Ldha	-0.299	0.009
Pgam1	-0.124	0.010
Hk1	0.213	0.016
Eno3	-0.479	0.017
Gapdh	-0.122	0.021
Pkm	-0.108	0.082
Gck	-0.427	0.110
Pfkp	0.114	0.172
Pklr	-0.733	0.385
Hk3	-0.281	0.421
Pfkl	-0.103	0.456
Pgam2	0.120	0.607
Aldob	0.237	0.673
Hk2	0.028	0.890
Eno2	0.008	0.912
Pfkm	-0.002	0.980

12.3.2 Glucose Transporter Genes

Among glucose transporter genes, Slc2a1, Slc2a6, and Slc2a8 showed reduced expression in brain tissue following hepatic ApoE4→ApoE2 conversion, while Slc2a3 displayed a mild increase. Other transporters, including Slc2a4, Slc5a1, and Slc2a2, did not show significant changes.

gene	log2FoldChange	padj
Slc2a6	-0.465	0.000
Slc2a1	-0.288	0.002
Slc2a8	-0.298	0.004
Slc2a3	0.174	0.080
Slc2a4	-0.182	0.355
Slc5a1	-0.241	0.431
Slc2a2	0.296	NA

12.3.3 Pentose Phosphate Pathway Genes

Within the pentose phosphate pathway, Tkt showed decreased expression in brain tissue following hepatic ApoE4→ApoE2 conversion. The remaining genes analyzed, Rpia, Pgd, and Rpe, did not display significant expression changes.

gene	log2FoldChange	padj
Tkt	-0.225	0.000
Rpia	0.136	0.214
Pgd	-0.013	0.879
Rpe	0.001	0.996

12.3.4 Pyruvate metabolism Genes

Lower Pdk2 and higher Pdp1 and Pdp2 expression reopen the pyruvate dehydrogenase complex, enabling glucose-derived pyruvate to enter the TCA cycle and support oxidative metabolism.

gene	log2FoldChange	padj
Pdp1	0.207	0.001
Pdk2	-0.193	0.001
Pdk3	0.184	0.001
Pck2	-0.318	0.002
Dld	0.115	0.007
Dlat	0.176	0.022
Pdp2	0.233	0.022
Pdhx	0.096	0.040
Pdha1	0.114	0.094
Pdk1	0.114	0.237
Pdk4	0.107	0.369
Pdhb	0.038	0.393
Pck1	1.001	NA

12.3.5 Lactate Transporter Genes

Lactate transporter genes reflected a rebalancing of the astrocyte–neuron metabolic coupling. The reduction of Slc16a3 (MCT4) and the increase of Slc16a7 (MCT2) suggest less lactate export from astrocytes and greater neuronal uptake, restoring a healthy lactate shuttle.

gene	log2FoldChange	padj
Slc16a7	0.436	0.002
Slc16a14	0.454	0.004
Slc16a3	-0.431	0.015
Slc16a8	-0.606	0.023
Slc16a11	-0.429	0.049
Slc16a10	0.234	0.101
Slc16a1	-0.012	0.936
Slc16a13	0.015	0.938

12.3.6 Glycogen Metabolism Genes

Regulators of glycogen metabolism, such as Pygm, Agl, and Phka2, also adjusted in line with this more stable metabolic state, showing decreased mobilization of glycogen stores.

gene	log2FoldChange	padj
Pygm	-0.235	0.010
Agl	0.164	0.035
Phka2	0.209	0.039
Gbe1	0.125	0.151
Phkg1	-0.109	0.383
Pygl	-0.107	0.423
Pygb	-0.034	0.650
Gys1	-0.043	0.653
Phka1	-0.022	0.828
Aga	-0.008	0.956
Gys2	-0.149	NA

12.3.7 Metabolic Regulators

12.3.8 11.3.7 Metabolic Regulators

Several metabolic regulators changed expression in brain tissue following hepatic ApoE4→ApoE2 conversion. Insr, Gsk3b, Prkaa2, Pik3ca, Akt3, Irs1, Pik3cb, and Prkaa1 were upregulated, while Akt1, Prkag1, Akt2, and Rheb were downregulated. Plin2 also decreased in expression. Other genes, including Mtor, Irs2, Prkab1, Stk11, Prkag2, Prkab2, and Gsk3a, showed minor or nonsignificant changes.

gene	log2FoldChange	padj
Insr	0.358	0.000
Gsk3b	0.453	0.000
Akt1	-0.215	0.000
Prkag1	-0.253	0.000
Akt2	-0.237	0.000
Prkaa2	0.348	0.001
Pik3ca	0.262	0.001
Akt3	0.289	0.002
Irs1	0.286	0.004
Pik3cb	0.216	0.007
Prkaa1	0.196	0.019
Plin2	-0.231	0.023
Depdc5	0.139	0.031
Rheb	-0.160	0.034
Mtor	0.148	0.061
Irs2	0.153	0.074
Prkab1	-0.154	0.093
Stk11	-0.050	0.352
Prkag2	0.068	0.500
Prkab2	0.053	0.689
Gsk3a	-0.024	0.690

12.3.9 Mitochondrial Transport Genes

Mitochondrial transporter genes (Mpc1, Mpc2, Slc25a1, Slc25a10) declined, indicating lower demand for emergency substrate import.

gene	log2FoldChange	padj
Slc25a10	-0.495	0.000
Slc25a1	-0.435	0.000
Slc25a11	-0.383	0.000
Mpc2	-0.276	0.000
Mpc1	-0.206	0.001

12.3.10 All metabolic pathways (only significant genes )

This section lists all genes with significant expression changes across major metabolic pathways in brain tissue after hepatic ApoE4→ApoE2 conversion.

id	gene	log2FoldChange	padj
glucose_transporters	Slc2a6	-0.465	0.000
glucose_transporters	Slc2a1	-0.288	0.002
glucose_transporters	Slc2a8	-0.298	0.004
glyco_core	Ldhb	-0.272	0.000
glyco_core	Aldoa	-0.354	0.000
glyco_core	Pgk1	-0.209	0.000
glyco_core	Eno1	-0.261	0.000
glyco_core	Aldoc	-0.237	0.002
glyco_core	Ldha	-0.299	0.009
glyco_core	Pgam1	-0.124	0.010
glyco_core	Hk1	0.213	0.016
glyco_core	Eno3	-0.479	0.017
glyco_core	Gapdh	-0.122	0.021
glycogen_metabolism	Pygm	-0.235	0.010
glycogen_metabolism	Agl	0.164	0.035
glycogen_metabolism	Phka2	0.209	0.039
lactate_transport	Slc16a7	0.436	0.002
lactate_transport	Slc16a14	0.454	0.004
lactate_transport	Slc16a3	-0.431	0.015
lactate_transport	Slc16a8	-0.606	0.023
lactate_transport	Slc16a11	-0.429	0.049
metabolic_regulators	Insr	0.358	0.000
metabolic_regulators	Gsk3b	0.453	0.000
metabolic_regulators	Akt1	-0.215	0.000
metabolic_regulators	Prkag1	-0.253	0.000
metabolic_regulators	Akt2	-0.237	0.000
metabolic_regulators	Prkaa2	0.348	0.001
metabolic_regulators	Pik3ca	0.262	0.001
metabolic_regulators	Akt3	0.289	0.002
metabolic_regulators	Irs1	0.286	0.004
metabolic_regulators	Pik3cb	0.216	0.007
metabolic_regulators	Prkaa1	0.196	0.019
metabolic_regulators	Plin2	-0.231	0.023
metabolic_regulators	Depdc5	0.139	0.031
metabolic_regulators	Rheb	-0.160	0.034
mitochondrial_transport	Slc25a10	-0.495	0.000
mitochondrial_transport	Slc25a1	-0.435	0.000
mitochondrial_transport	Slc25a11	-0.383	0.000
mitochondrial_transport	Mpc2	-0.276	0.000
mitochondrial_transport	Mpc1	-0.206	0.001
pentose_phosphate_pathway	Tkt	-0.225	0.000
pyruvate_metabolism	Pdp1	0.207	0.001
pyruvate_metabolism	Pdk2	-0.193	0.001
pyruvate_metabolism	Pdk3	0.184	0.001
pyruvate_metabolism	Pck2	-0.318	0.002
pyruvate_metabolism	Dld	0.115	0.007
pyruvate_metabolism	Dlat	0.176	0.022
pyruvate_metabolism	Pdp2	0.233	0.022
pyruvate_metabolism	Pdhx	0.096	0.040

The data show that the ApoE4 mouse brain lives under chronic metabolic stress, a condition reversed by hepatic ApoE2 expression. The ApoE4 state is marked by glucose hypometabolism, which forces astrocytes to accumulate small lipid droplets stabilized by high Plin2 expression. This is not a resting phase but an inefficient, oxygen-hungry state where fatty acids are oxidized incompletely, producing metabolic intermediates (Farmer, Kluemper, and Johnson 2019). An accompanying rise in Slc2a1, Slc2a6, Slc2a8, Mpc1, Mpc2, Slc25a1, and Slc25a10 may reflect a failed attempt to compensate for the energy shortage. Hepatic ApoE2 expression restores balance, normalizing these transport systems.

This correction involves a deeper reprogramming of glucose metabolism. In ApoE4 brains, glycolysis runs fast but inefficiently, shunting pyruvate to lactate through Ldha and the astrocytic exporter Slc16a3 (MCT4). With hepatic ApoE2, Hk1 rises, trapping glucose in cells, while glycolytic enzymes such as Aldoa, Pgk1, and Eno1 fall. At the same time, Pdk2 declines and Pdp1 and Pdp2 increase, reopening the pyruvate dehydrogenase complex and steering metabolism toward aerobic respiration. This shift calms glycolytic activity and reduces glycogen breakdown, shown by lower Pygm expression.

The lactate shuttle also resets. Astrocytic lactate export drops with reduced Ldha and Slc16a3, while neuronal uptake improves through Slc16a7 (MCT2). The fall in Tkt suggests that oxidative stress decreases as the pentose phosphate pathway relaxes.

Finally, key regulators of energy balance respond. Insr and Irs1 rise, restoring insulin sensitivity, while Plin2 declines, signaling relief from lipid stress. The downregulation of oxidative phosphorylation genes confirms that the brain’s metabolism has cooled from a high-output, inefficient state to a steady, aerobic equilibrium. In sum, hepatic ApoE2 expression resolves the metabolic strain of ApoE4, returning the brain to an efficient and balanced homeostasis.

Farmer, Brandon, Jude Kluemper, and Lance Johnson. 2019. “Apolipoprotein E4 Alters Astrocyte Fatty Acid Metabolism and Lipid Droplet Formation.” Cells 8 (2): 182. https://doi.org/10.3390/cells8020182.